ABSTRACT Cytotoxic T lymphocytes (CTLs) are a key element of adaptive immunity against intracellular infections and cancers. They are potent antigen-directed killer cells that specifically eliminate infected or diseased target cells. Following pathogen control, a subset of effector CTLs gradually shutdown their immediate cytotoxic functions, while retaining heightened responsiveness to future encounter with the same pathogen. Long-term persistence of quiescent CTL memory sentinels is deemed a highly desirable outcome for vaccines and modern adoptive cell immunotherapies, to maintain durable protective immunity. The goal of this proposal is to gain mechanistic insight into how functionally active effector CTLs convert into quiescent memory. The classic paradigm is that effector-to-memory conversion is the default differentiation pathway for memory precursor effector cells (MPECs) after antigen clearance. This is believed to occur simply because of loss of T cell receptor (TCR) stimulation. However, recent studies from our group and others show that Tregs exert a critical role in the process of effector-to-memory transition following antigen clearance. Tregs promote the development of quiescent, yet functionally poised memory cells by suppressing effector and proliferative programs in memory-fated CTLs. In the absence of Tregs during effector-to-memory transition, critical metabolic and transcriptional remodeling does not occur, thus leading to memory failure. Based on brightest expression levels of CTLA-4 on Tregs (amongst all immune cells), and recent human studies noting dysregulation of Tregs and hyperactivation of T and B cells in CTLA-4 haploinsufficient individuals, we propose that CTLA-4 is a critical mediator of Treg-dependent reprogramming of MPECs from effector-to-memory state. We further hypothesize that CTLA-4 mediates its effects through direct as well indirect effects on CD8 T cells. Our preliminary studies showing that exogenous administration of CTLA-4 restores homeostasis and fully reverses the defects in CD8 T cell memory associated with Treg ablation, lend support to the notion that CTLA- 4 is the primary initiating signal for Treg-mediated memory CD8 T cell quiescence. Using unique mouse models, and high throughput cellular and biochemical readouts, the goal of this proposal is to rigorously investigate the interplay of cellular networks, and transcriptional and metabolic programs in orchestrating Treg- mediated help during memory CD8 T cell development. Over the last decade, there has been significant progress in understanding when and how memory fate commitment occurs. However, less is known about how quiescent memory cells are formed from effector CTLs once antigen is cleared, and how quiescent memory cells are maintained for life. Successful completion of proposed studies will uncover novel strategies for augmenting CD8 T cell memory during immunization, and will also afford newer immunotherapeutic approaches against cancers and for maintaining homeostasis in autoimmune disorders.